Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A Analysis of the Bioplex 3.0 interactome identified 42 potential DLK1 binding partners. The pie chart shows the fractions of candidate proteins containing extracellular domains (magenta) and intracellular proteins (teal). ACVR2B (bold) is the only known cell surface receptor among extracellular domain-containing proteins. B Confocal microscopy images depicting wild type U2OS cells or DLK1-expressing U2OS cells stained with recombinant ACVR2B-Fc protein. ACVR2B-Fc binding was detected with an anti-Fc Alexa Fluor 488 antibody, the contours of the cells were visualized by actin staining (magenta) using phalloidin 647, and nuclei were counterstained using DAPI (blue). The images are represented as maximum projections of 5 z-slices taken 0.8 µm apart. Scale, 20 µm. The experiment was independently repeated three times. C SPR was used to determine the steady-state binding affinity between DLK1(N-EGF6) and ACVR2B-Fc. The DLK1 protein was injected over a sensor chip containing immobilized ACVR2B-Fc and the data was fitted to a 1:1 binding model. RU = resonance units. The associated SPR sensograms for this data are shown in Supplementary Fig. . D Confocal microscopy images depicting the staining of U2OS cells overexpressing ACVR2B coupled to a GFP SPARK (green) tag, with DLK1-Fc protein (magenta). DLK1 binding was detected using an anti-Fc Alexa Fluor 647 antibody. Nuclei counterstained with DAPI (blue). Scale, 20 µm. The experiment was independently repeated two times. E Flow cytometry histograms depicting the binding of DLK1-Fc protein to yeast expressing ACVR2B and eleven other TGF-β superfamily receptors. The experiment was independently repeated two times. Source data are provided as a Source Data file.

Article Snippet: ACVR2B pCMV3-C-GFPSpark or pCMV3-C-GFPSpark control vector (SinoBiological, Cat# HG10229-ACG and Cat# CV026) were transfected using Lipofectamine 3000 in U2OS cells and stable cell lines were selected using 200 μg/ml hygromycin.

Techniques: Binding Assay, Cell Surface Receptor Assay, Confocal Microscopy, Expressing, Staining, Recombinant, Injection, Flow Cytometry

Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A Crystal structure of DLK1 domains EGF5-6 (magenta) in complex with the extracellular domain of ACVR2B (teal). B A surface model of ACVR2B (teal) with DLK1(magenta) overlaid with a cartoon representation of myostatin (yellow). C Zoom in panel showing Trp 78 and Phe 101 of ACVR2B forming hydrophobic interactions with Arg 193 of DLK1. D Zoom in panel showing Phe 82 of ACVR2B packing against Val 209 and Val 229 of DLK1, and Arg 56 of ACVR2B forming a hydrogen bond with Gln 228 . E SPR isotherms comparing the binding between DLK1(EGF5-6) or DLK1(EGF5-6) R193D -mutant to ACVR2B-Fc. The DLK1 proteins were injected over a sensor chip containing immobilized ACVR2B-Fc and the data was fitted to a 1:1 binding model. RU = resonance units. F SPR isotherms comparing the binding between DLK1 and three ACVR2B-Fc interface mutants. The R56A mutation in ACVR2B was associated with a ~ 6-fold decrease in DLK1-binding affinity compared to WT ACVR2B, and there was a complete loss of DLK1 binding to the W78A or F101A mutants. RU = resonance units. Source data are provided as a Source Data file.

Article Snippet: ACVR2B pCMV3-C-GFPSpark or pCMV3-C-GFPSpark control vector (SinoBiological, Cat# HG10229-ACG and Cat# CV026) were transfected using Lipofectamine 3000 in U2OS cells and stable cell lines were selected using 200 μg/ml hygromycin.

Techniques: Binding Assay, Mutagenesis, Injection

Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A The structure of ACVR2A (PDB ID: 5NH3 ) was superimposed onto ACVR2B in the DLK1-ACVR2B complex structure (PDB ID: 9D20 ). B A zoom window shows Phe 82 of ACVR2B inserted into the pocket formed by DLK1 residues Val 209 and Val 229 . The analogous residue of ACVR2A, Ile 83 , is not predicted to fit into this pocket. C A zoom window shows Arg 193 of DLK1 forming polar interactions with Thr 93 and Glu 94 of ACVR2B. The analogous residues in ACVR2A, Lys 94 and Lys 95 , are not predicted to form charge complementary interactions. D–F Electrostatic potential surface representation of the DLK1-ACVR2B complex. The arrow indicates the ACVR2B interface glutamate (E94) residue that is substituted for a lysine (K95) in ACVR2A. D ACVR2B alone ( E ) and ACVR2A alone ( F ). G Sequence alignment of human ACVR2B and ACVR2A. Selected conserved (teal) and non-conserved (yellow) residues forming the DLK1-ACVR2B interface are highlighted. The E94 residue of ACVR2B is indicated with a black arrow.

Article Snippet: ACVR2B pCMV3-C-GFPSpark or pCMV3-C-GFPSpark control vector (SinoBiological, Cat# HG10229-ACG and Cat# CV026) were transfected using Lipofectamine 3000 in U2OS cells and stable cell lines were selected using 200 μg/ml hygromycin.

Techniques: Residue, Sequencing

Journal: Nature Communications

Article Title: Molecular mechanism of Activin receptor inhibition by DLK1

doi: 10.1038/s41467-025-60634-3

Figure Lengend Snippet: A Illustration of Myostatin-ACVR2B signaling in the presence or absence of DLK1. Binding of the canonical ligand Myostatin to ACVR2B and ACVR1B leads to the formation of a 2:2:2 complex and subsequent activation of SMAD2/3 (left). The EGF5 domain of DLK1 binds to ACVR2B to inhibit ligand signaling (right), and the DLK1(EGF5-6) region used for co-crystallization is indicated with a dashed circle. Created in BioRender. Antfolk, D. (2025) https://BioRender.com/p73c456 B DLK1 inhibits Myostatin-ACVR2B signaling in a HEK293-(CAGA) 12 reporter assay. Myostatin treatment at 2 nM is represented as 100% activation, and a decrease in activation was observed upon treatment with increasing concentrations (125 nM-16 µM) of soluble DLK1. Data is represented as normalized relative luciferase units (RLU) represented as the mean of triplicate wells from one representative experiment. The experiment was independently repeated three times. C DLK1 transfected into HEK293-(CAGA) 12 reporter cells inhibit Myostatin signaling. Data is represented as RLU based on quadruplicate wells from one representative experiment. The experiment was independently repeated two times. D Representative microscopy images showing C2C12 myoblast differentiation in the presence of Myostatin, Myostatin + DLK1, or Myostatin + DLK1 R193D (loss-of-ACVR2B-binding mutant). Control cells were allowed to differentiate for 72 h. Myostatin treatment (4 ug/ml) inhibits C2C12 myoblast differentiation into myotubes as determined by MyoHC staining. C2C12 cells were fixed with 4% PFA, immunostained with an anti-MyoHC antibody and an anti-mouse IgG Alexa Fluor 488 secondary antibody. Nuclei were counterstained with Hoechst 33342. Nuclei represented with pseudo color (magenta) in zoom in panels. Scale bar, 100 μm. The experiment was independently repeated four times. Source data are provided as a Source Data file.

Article Snippet: ACVR2B pCMV3-C-GFPSpark or pCMV3-C-GFPSpark control vector (SinoBiological, Cat# HG10229-ACG and Cat# CV026) were transfected using Lipofectamine 3000 in U2OS cells and stable cell lines were selected using 200 μg/ml hygromycin.

Techniques: Binding Assay, Activation Assay, Crystallization Assay, Reporter Assay, Luciferase, Transfection, Microscopy, Mutagenesis, Control, Staining